In the early 1960's, zymosan, a crude insoluble yeast extract prepared by boiling yeast before and after trypsin treatment, was noted to produce marked hyperplasia and functional stimulation of the reitculoendothelial system in rodents. In animal studies, zymosan preparations were shown to inactivate complement component C3, to enhance antibody formation, to promote survival following irradiation, to increase resistance to bacterial infections, to inhibit tumor development, to promote graft rejection, and to inhibit dietary-induced hypercholesterolemia and cholesterosis. Zymosan was shown to consist of polysaccharides, proteins, fats, and inorganic elements; however, subsequent studies identified the active components of the yeast cell wall as a pure polysaccharide, specifically .beta.-glucan. Repetition of biological assays with .beta.-glucan indicated that most of the above functional activities identified with zymosan were retained by the purified .beta.-glucan preparation.
The properties of .beta.-glucan are quite similar to those of endotoxin in increasing nonspecific immunity and resistance to infection. The activities of .beta.-glucan as an immune adjuvant and hemopoietic stimulator compare to those of more complex biological response modifiers (BRMs), such as bacillus Calmette-Guerin (BCG) and Corynebacterium parvum. The functional activities of yeast .beta.-glucan are also comparable to those structurally similar carbohydrate polymers isolated from fungi and plants. These higher molecular weight (1-3)-.beta.-D-glucans such as schizophyllan, lentinan, krestin, grifolan, and pachyman exhibit similar immunomodulatory activities. A common mechanism shared by all these .beta.-glucan preparations is their stimulation of cytokines such as interleukin-1 and TNF. Lentinan has been extensively investigated for its antitumor properties, both in animal models at 1 mg/kg for 10 days and in clinical trials since the late 1970s in Japan for advanced or recurrent malignant lymphoma and colorectal, mammary, lung and gastric cancers. In cancer chemotherapy, lentinan has been administered at 0.5-5 mg/day, I. M. or I. V., two or three times per week alone, or in combination with antineoplastic drugs. In addition to the activities ascribed to yeast glucans, studies suggest lentinan acts as a T-cell immunopotentiator, inducing cytotoxic activities, including production of interleukin-1 (IL-1), colony-stimulating factor (CSF) and interleukin-3 (IL-3). (Chihara al., 1989, Int. J. Immunotherapy, 4:145-154; Hamuro and Chihara, In Lentinan, An Immunopotentiator)
Various preparations of both particulate and soluble .beta.-glucans have been tested in animal models to specify biological activities. The use of soluble and insoluble .beta.-glucans alone or as vaccine adjuvants for viral and bacterial antigens has been shown in animal models to markedly increase resistance to a variety of bacterial, fungal, protozoan and viral infections. The hemopoietic effects of .beta.-glucan have been related to increased peripheral blood leukocyte counts and bone marrow and splenic cellularity, reflecting increased numbers of granulocyte-macrophage progenitor cells, splenic pluripotent stem cells, and erythroid progenitor cells, as well as, increased serum levels of granulocyte-monocyte colony-stimulating factor (GM-CSF). Furthermore, the hemopoietic and anti-infective effects of .beta.-glucan were active in cyclophosphamide-treated immunosuppressed animals. .beta.-glucan was shown to be beneficial in animal models for trauma, wound healing and tumorigenesis. However, various insoluble and soluble preparations of .beta.-glucan differed significantly in biological specificity and potency, with effective dosages varying from 25 to 500 mg/kg I. V. or I. P. in models for protection against infection and for hemopoiesis. Insoluble preparations demonstrated undesirable toxicological properties manifested by hepatosplenomegaly and granuloma formation. Clinical interest focused on a soluble glucan preparation which would retain biological activity yet yield negligible toxicity when administered systemically. Chronic systemic administration of a soluble phosphorylated glucan over a wide range of doses (40-1000 mg/kg) yielded negligible toxicity in animals (DiLuzio et al, 1979, Int. J. of Cancer, 24:773-779; DiLuzio, U.S. Patent 4,739,046).
The molecular mechanism of action of .beta.-glucan has been elucidated by the demonstration of specific .beta.-glucan receptor binding sites on the cell membranes of human neutrophils and macrophages. Mannans, galactans, .alpha.(1-4)linked glucose polymers and .beta.(1-4)-linked glucose polymers have no avidity for this receptor. These .beta.-glucan binding sites are opsonin-independent phagocytic receptors for particulate activators of the alternate complement pathway, similar to Escherichia coli lipopolysaccharide (LPS), inulin and some animal red blood cells. Ligand binding to the .beta.-glucan receptor, in the absence of antibody, results in complement activation, phagocytosis, lysosomal enzyme release, and prostaglandin, thromboxane and leukotriene generation; thereby increasing nonspecific resistance to infection. However, all of the soluble .beta.-glucan preparations described in the prior art demonstrated stimulation of cytokines. Increases in plasma and splenic levels of interleukins 1 and 2 (IL-1, IL-2) in addition to tumor necrosis factor (TNF) were observed in vivo and corresponded to induction of synthesis of these cytokines in vitro. See Sherwood et al., 1987, Int. J. Immunopharmac., 9:261-267 (enhancement of IL-1 and IL-2 levels in rats injected with soluble glucan); Williams et al., 1988, Int. J. Immunopharmac., 10:405-414 (systemic administration of soluble glucan to AIDS patients increased IL-1 and IL-2 levels which were accompanied by chills and fever); Browder et al., 1990, Ann. Surg., 211:605-613 (glucan administration to trauma patients increased serum IL-1 levels, but not TNF levels); Adachi et al., 1990, Chem. Pharm. Bull., 38:988-992 (chemically crosslinked .beta.(1-3) glucans induce IL-1 production in mice).
Interleukin-1 (IL-1) is a primary immunologic mediator involved in cellular defense mechanisms. Numerous studies have been carried out on the application of IL-1 to enhance non-specific resistance to infection in a variety of clinical states. Pomposelli et a., J. Parent. Ent. Nutr., 12(2):212-218, (1988). The major problem associated with the excessive stimulation or exogenous administration of IL-1 and other cellular mediators in humans is toxicity and side effects resulting from the disruption of the gentle balance of the immunoregulatory network. Fauci et al., Anals. of Internal Medicine, 106:421-433 (1987). IL-1 is an inflammatory cytokine that has been shown to adversely affect a variety of tissues and organs. For instance, recombinant IL-1 has been shown to cause death, hypotensive shock, leukopenia, thrombocytopenia, anemia and lactic acidosis. In addition, IL-1 induces sodium excretion, anorexia, slow wave sleep, bone resorption, decreased pain threshold and expression of many inflammatory-associated cytokines. It is also toxic to insulin secreting beta cells. Patients suffering from a number of inflammatory diseases already have elevated levels of IL-1 in their systems. Administration of agents that enhance further IL-1 production only exacerbate these inflammatory conditions. Thus, it would be beneficial to develop an agent that only selectively stimulates the immune system but which does not stimulate IL-1 or TNF.
Tumor necrosis factor (TNF) is also involved in infection, inflammation and cancer. Small amounts of TNF release growth factors while in larger amounts, TNF can cause septic shock, aches, pains, fever, clot blood, degrade bone and stimulates white blood cells and other immune defenses. Development of a drug which minimizes these adverse side affects caused by the release of TNF would be highly desirable, especially for individuals whose immune system has been activated due to infection, autoimmune disease or cancer.